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Role of Tet proteins in epigenetic regulation of embryonic stem cell biology

Tet 蛋白在胚胎干细胞生物学表观遗传调控中的作用

基本信息

批准号：
10294252
负责人：
Meelad Dawlaty
金额：
$ 35.07万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-12-05 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10294252
关键词：
Address Affect Biological Biological Assay Biology Cell Cycle Progression Cell Differentiation process Chromatin Complex DNA Data Deacetylation Development Disease Embryo Embryonic Development Enzymes Epigenetic Process Family Future Gene Activation Gene Expression Gene Expression Regulation Gene Silencing Genes Genome Genomics Goals Growth Health Human In Vitro Knock-out Knockout Mice Lead Malignant Neoplasms Mediating Modification Molecular Mus Nucleic Acid Regulatory Sequences Outcome Oxides Phenotype Physiological Pluripotent Stem Cells Property Protein translocation Proteins Regenerative Medicine Regulation Regulator Genes Regulatory Element Research Role Stem Cell Development Testing Therapeutic Work comparative demethylation differential expression embryonic stem cell epigenetic marker epigenetic regulation epigenome epigenomics gene repression induced pluripotent stem cell mutant novel novel marker overexpression programs promoter public health relevance recruit stem cell biology stem cell proliferation stem cell therapy stem cells transcriptomics

项目摘要

Modified Project Summary/Abstract Section: The main goal of this project is to dissect gene regulatory functions of Tet enzymes in ESCs, and define the significance of noncanonical (non-enzymatic) roles of these proteins in gene repression and in stem cell biology. The Ten-eleven-translocation (Tet) enzymes (Tet1, Tet2, Tet3) regulate gene expression program in ESCs and during development. They facilitate DNA demethylation by oxidizing 5-methylcytosine (5mC) to 5- hydroxymethylcytosine and other derivatives. The enzymatic function of Tet enzymes in DNA demethylation and its significance in ESCs and development has been well investigated as part of my prior work in the field. However, recent findings from my lab strongly suggest that key biological roles of Tet proteins are independent of their catalytic activity. We have found that more genes are differentially expressed in Tet1 knockout ESCs than in Tet1 catalytic mutant ESCs (vs. wild type ESCs). Majority of these genes are overexpressed, suggesting that noncatalytic functions of Tet enzymes regulate gene repression in ESCs and contribute to their biology. Consistently, we found that catalytically inactive Tet enzymes rescued some of the phenotypes of Tet deficient ESCs. These observations build the initial premise for this project. We hypothesize that in addition to their DNA demethylase activity, Tet enzymes regulate the epigenome independent of their enzymatic activity. This involves recruiting chromatin repressive complexes to gene regulatory regions for proper gene repression. This constitutes a novel layer of epigenetic regulation that influences key features of ESC biology including proliferation as well as in vitro differentiation and lineage specification. Using a recently developed platform in my lab that consists of Tet catalytic mutant and knockout ESCs, we will apply molecular, cellular and developmental approaches to dissect the catalytic dependent and independent requirements of Tet enzymes in stem cells, and establish the significance of noncanonical roles of Tet enzymes in ESC biology. We propose the following aims: (1) We will establish the noncanonical roles of Tet enzymes in recruiting chromatin repressive complexes, Sin3a and PRC2, to gene regulatory elements for gene silencing. (2) We will define the biological relevance of Tet-mediated noncanonical gene repression in ESC proliferation, as well as in in vitro endodermal and trophectodermal differentiation and lineage specification. Our findings will reveal novel epigenetic mechanisms of regulation of ESCs by Tet enzymes. This will enhance our understanding of the epigenetic networks in stem cells and will open future opportunities for investigating Tet-mediated gene regulation during development and in diseases.

修改后的项目摘要/摘要部分：本项目的主要目标是剖析胚胎干细胞中Tet酶的基因调控功能，并确定这些蛋白在基因抑制和干细胞生物学中的非规范(非酶)作用的意义。Ten-11易位(Tet)酶(Tet1、TET2、Tet3)调节胚胎干细胞和发育过程中的基因表达程序。它们通过将5-甲基胞嘧啶(5mC)氧化成5-羟甲基胞嘧啶和其他衍生物来促进DNA去甲基化。作为我在该领域前期工作的一部分，Tet酶在DNA去甲基化中的酶功能及其在ESCs和发育中的意义已经得到了很好的研究。然而，我的实验室最近的发现有力地表明，Tet蛋白的关键生物学作用与它们的催化活性无关。我们发现，在Tet1基因敲除的ESCs中，差异表达的基因比在Tet1催化突变的ESCs(与野生型ESCs)中的基因更多。这些基因中的大多数都是过表达的，这表明Tet酶的非催化功能调节了ESCs的基因抑制，并对其生物学做出了贡献。我们一致地发现，催化失活的Tet酶挽救了Tet缺陷ESCs的一些表型。这些观察结果为本项目奠定了最初的前提。我们推测，除了DNA脱甲基酶活性外，Tet酶还独立于其酶活性调节表观基因组。这涉及到将染色质抑制复合体招募到基因调控区域，以进行适当的基因抑制。这构成了一个新的表观遗传调节层，影响了ESC生物学的关键特征，包括增殖以及体外分化和谱系规范。利用我实验室最近开发的一个由Tet催化突变和敲除ESCs组成的平台，我们将应用分子、细胞和发育方法来剖析干细胞中Tet酶的催化依赖和独立需求，并建立Tet酶在ESC生物学中的非规范作用的意义。我们提出了以下目标：(1)我们将确定Tet酶在招募染色质抑制复合体Sin3a和PrC2到基因调控元件中的非典型作用，以实现基因沉默。(2)我们将确定Tet介导的非规范基因抑制在ESC增殖中的生物学意义，以及在体外内胚层和滋养外胚层分化和谱系分化中的生物学意义。我们的发现将揭示Tet酶调控ESCs的新的表观遗传学机制。这将增强我们对干细胞表观遗传网络的理解，并将为研究Tet在发育过程中和疾病中的基因调控打开未来的机会。